The present invention relates generally to the field of virology. More particularly, the invention relates to the discovery that peptides that bind to the hepatitis B virus (HBV) core and e antigens can be used to inhibit HBV infection.
Of the many viral causes of human hepatitis, few are of greater global importance than hepatitis B virus (HBV). Approximately 300 million people worldwide are chronically infected and some of these chronically infected individuals develop severe pathologic consequences including chronic hepatic insufficiency, cirrhosis, and hepatocellular carcinoma (HCC). (See Fields Virology, third ed., edited by Fields et al., Lipponcott-Raven Publishers, Philidelphia 1996 pp. 2703 and Lee et al., Cancer, 72:2564-7 (1993)). Primary infection may be asymptomatic (e.g,, in chronically infected individuals) or may result in varying degrees of acute liver injury. (Milich et al., Springer Seminars in Immunopathology, 17:149-66 (1995)).
HBV is unusual among animal viruses in that infected cells produce multiple types of virus-related particles. (See Fields Virology, third ed., edited by Fields et al., Lipponcott-Raven Publishers, Philidelphia 1996 pp. 2704). Electron microscopy of partially purified preparations of HBV shows three types of particles, a 42-47 nm infectious particle (referred to as xe2x80x9cDane particlesxe2x80x9d), non-infectious 20 nm spheres, and non-infectious 20 nm diameter filaments of variable length. Id. at 2705-2705. The HBV genome encodes at least five structural proteins: the envelope or surface proteins preS 1, preS2, and S (HBsAg); the polymerase; and the core or capsid antigen (HBcAg). All three forms of HBV particles have HBsAg, which serves as an epitope for neutralizing antibodies and is the basis for state of the art HBV diagnostics. In contrast, only the Dane particles have HBcAg, a 21 kD phosphoprotein that is believed to be phosphorylated in vivo. Id. at 2705. The HBV genome also encodes the non-structural proteins HBeAg and X. The HBcAg and the HBeAg are translated from two different mRNAs that are transcribed from the same open reading frame. The longer of the two mRNAs encodes HBeAg. HBcAg and the HBeAg share an amino acid sequence of approximately 150 residues.
HBcAg is highly immunogenic in humans and mice. Investigators have observed that HBcAg induces B-cells to produce IgM and, thus, is currently classified as a partially T cell independent antigen. (Milich and McLachlan, Science, 234:1398-401 (1986)). HBcAg can also crosslink B-cell surface receptors and membrane bound IgM on naive B-cells and, in turn, HBcAg can be taken up, processed, and presented to HBcAg-specific CD4xe2x88x92T cells. (Milich et al., Proc Natl Acad Sci USA, 94:14648-14653 (1997)). Quite surprisingly, B-cells that are able to bind and present HBcAg exist in great numbers in naive non-immunized mice. The identification of molecules that inhibit HBV infection by interacting with HBcAg and/or HBeAg remains a largely unrealized goal.
The invention described herein concerns the identification and manufacture of molecules that interact with HBcAg and/or HBeAg and thereby inhibit HBV infection or modulate a host immune system response or both. Molecules that interact with HBcAg and/or HBeAg, also referred to as xe2x80x9cbinding partnersxe2x80x9d, are designed from fragments of antibodies and other proteins that interact with HBcAg and/or HBeAg. Accordingly, an amino acid sequence corresponding to the binding domains of monoclonal or polyclonal antibodies or proteins that bind HBcAg and/or HBeAg is used as a template for the design of synthetic molecules, including but not limited to, peptides, derivative or modified peptides, peptidomimetics, and chemicals. A preferred binding partner, for example, is a molecule called a xe2x80x9cspecificity exchangerxe2x80x9d, which comprises a first domain that interacts with HBcAg and/or HBeAg and a second domain that has an epitope for a high titer antibody, preferably an epitope on a pathogen or a toxin. The binding partners described herein can be manufactured by conventional techniques in peptide chemistry and/or organic chemistry.
Methods to characterize binding partners are also embodiments. The term xe2x80x9ccharacterization assayxe2x80x9d is used to refer to an experiment or evaluation of the ability of a candidate binding partner and/or binding partner to interact with HBcAg and/or HBeAg, inhibit HBV infection, or modulate a host immune response. Some characterization assays, for example, evaluate the ability of a binding partner to bind to a multimeric agent having HBcAg and/or HBeAg disposed thereon or vice versa. Other characterization assays access the ability of a binding partner to fix complement and/or bind to a high titer antibody. Additional characterization assays determine whether a binding partner can effect viral infection in cultured cell lines or infected animals. Still further, some embodiments evaluate the ability of a binding partner to modulate a host immune system response, as measured by cytokine production and/or T cell proliferation.
Binding partners can be used as immunochemicals for the detection of HBcAg and/or HBeAg and can be incorporated into diagnostic methods and kits. Binding partners, preferably specificity exchangers, can also be incorporated into pharmaceuticals and used to treat or prevent HBV infection. A preferred embodiment concerns a method of treating or preventing HBV infection by identifying a subject in need and administering said subject a therapeutically effective amount of binding partner.
As described herein, embodiments include a peptide that binds HBcAg or HBeAg having about 3-50 amino acids residues. Preferably, the sequence of said peptide is selected from the group consisting of SEQ. ID. Nos. 4-45, 53, 54, 66-69, 71, and 74. Other embodiments include a peptide comprising the sequence of at least one of SEQ. ID. Nos. 1-3, a peptide comprising the sequence of SEQ. ID. No. 45, a peptide comprising the sequence of SEQ. ID. No. 54, a peptide comprising the sequence of SEQ. ID. No. 74, and a peptide having a specificity domain, which binds HBcAg or HBeAg and an antigenic domain joined to the specificity domain, wherein said antigenic domain binds a high titer antibody, preferably an epitope for a pathogen or toxin.
Related embodiments concern a peptidomimetic that corresponds to a peptide selected from the group consisting of SEQ. ID. No. 1, 2, 3, 45, 54, and 74 and an isolated or purified peptide that is less than 50 amino acids in length having the formula: X1nCKASX2n, wherein xe2x80x9cX1xe2x80x9d and xe2x80x9cX2xe2x80x9d are any amino acid and xe2x80x9cnxe2x80x9d is any integer, and wherein the molecule specifically binds HBcAg and/or HBeAg. Another way of describing the molecules of this class is by the formula: xe2x80x9cX1nCZASX2nxe2x80x9d, wherein: xe2x80x9cX1xe2x80x9d and xe2x80x9cX2xe2x80x9d are any amino acid and xe2x80x9cnxe2x80x9d is any integer, xe2x80x9cCxe2x80x9d is cysteine, xe2x80x9cZxe2x80x9d is lysine or argininexe2x80x9d, xe2x80x9cAxe2x80x9d is alanine, and xe2x80x9cSxe2x80x9d is serine. In some embodiments, the xe2x80x9cX1nxe2x80x9d or xe2x80x9cX2nxe2x80x9d encodes an epitope that binds a high titer antibody (e.g., an epitope on a pathogen or a toxin). Other embodiments include an isolated or purified peptide that is less than 50 amino acids in length having the formula: X1nCRASX2n, wherein xe2x80x9cX1xe2x80x9d and xe2x80x9cX2xe2x80x9d are any amino acid and xe2x80x9cnxe2x80x9d is any integer, and wherein the molecule specifically binds HBcAg and/or HBeAg. As above, another way of describing the molecules of this class is by the formula: xe2x80x9cX1nCZASX2nxe2x80x9d, wherein: xe2x80x9cX1xe2x80x9d and xe2x80x9cX2xe2x80x9d are any amino acid and xe2x80x9cnxe2x80x9d is any integer, xe2x80x9cCxe2x80x9d is cysteine, xe2x80x9cZxe2x80x9d is lysine or argininexe2x80x9d, xe2x80x9cAxe2x80x9d is alanine, and xe2x80x9cSxe2x80x9d is serine. In some embodiments, xe2x80x9cX1nxe2x80x9d or xe2x80x9cX2nxe2x80x9d encodes an epitope that binds a high titer antibody. Additional embodiments include a nucleic acid encoding a peptide selected from the group consisting of SEQ. ID. Nos. 1, 2, 3, 45, 54, and 74.
Some embodiments include a method of making a binding partner that interacts with HBcAg or HBeAg. By one approach, a region of a polypeptide that interacts with HBcAg or HBeAg is identified, the sequence of said region of the polypeptide is determined, and a synthetic or recombinant binding partner that corresponds to the sequence of said region of the polypeptide is produced. In some aspects of this embodiment, the polypeptide is an antibody and, in other aspects, the binding partner is a specificity exchanger. More embodiments include methods of making a pharmaceutical. By one approach, a binding partner that interacts with HBcAg or HBeAg is identified and a therapeutically effective amount of said binding partner is incorporated into a pharmaceutical. In preferred aspects of this method, the binding partner has a sequence selected from the group consisting of SEQ. ID. Nos. 4-45, 53, 54, 66-69, 71, and 74. Another method described herein concerns an approach to treat or prevent HBV infection. Accordingly, a subject in need of a molecule that inhibits HBV infection is identified and said subject is provided a binding partner that interacts with HBcAg or HBeAg, or both. Preferred aspects of this method involve a binding partner that has a sequence selected from the group consisting of SEQ. ID. Nos. 4-45, 53, 54, 66-69, 71, and 74.
Methods of identifying a binding partner that interacts with HBcAg or HBeAg are also embodiments. By one approach, a support comprising HBcAg or HBeAg is provided, the support is contacted with a candidate binding partner, and a biological complex comprising HBcAg or HBeAg and the candidate binding partner is detected, wherein detection of such complex indicates that said candidate binding partner is a binding partner interacts with HBcAg or HBeAg. In preferred aspects of this embodiment, the candidate binding partner has an amino acid sequence selected from the group consisting of SEQ. ID. Nos. 1-78. Another method of identifying a binding partner that inhibits HBV infection involves providing a cell that is infected with HBV, contacting said cell with a candidate binding partner, and identifying said binding partner when the presence of said candidate binding partner with said cell is associated with a decrease in HBV infection.
Furthermore, methods are provided that identify a binding partner that modulates an immune system response. Accordingly, one method is practiced by providing a naive antigen presenting cell, contacting said naive antigen presenting cell with a binding partner and a T cell that reacts to HBcAg or HBeAg, and detecting an inhibition or enhancement of T cell stimulation. In some embodiments, the detection step is performed by evaluating a change in cytokine production or T cell proliferation.
In another embodiment, a computerized system for identifying a binding partner that interacts with HBcAg or HBeAg is provided. This system includes a first data base comprising protein models of HBcAg or HBeAg; a second data base comprising the composition of a plurality of candidate binding partners; a search program that compares the protein model of the first data base with the compositions of the candidate binding partners of the second database; and a retrieval program that identifies a binding partner that interacts with the protein model of the first database. In some aspects of this embodiment, the candidate binding partners have an amino acid sequence selected from the group consisting of SEQ. ID. Nos. 1-78.
Additionally, a computer-based system for identifying a candidate binding partner having homology to a binding partner is provided. This system has a database with at least one of the sequences of SEQ ID NOS: 1-78 or a representative fragment thereof, a search program that compares a sequence of a candidate binding partner to sequences in the database to identify homologous sequence(s), and a retrieval program that obtains said homologous sequence(s).
A method of determining the presence of HBV in a biological sample is also an embodiment. This method is practiced by providing a biological sample, providing a binding partner that binds to HBcAg and/or HBeAg, wherein said binding partner has a sequence selected from the group consisting of SEQ. ID. Nos. 4-45, 53, 54, 66-69, 71, and 74, and determining the presence of HBV in the biological sample by monitoring whether said binding partner binds to HBcAg and/or HBeAg. Diagnostic kits for the detection of HBV infection are embodiments, as well. One such kit has a binding partner, wherein said binding partner has a sequence selected from the group consisting of SEQ. ID. Nos. 4-45, 53, 54, 66-69, 71, and 74.
The disclosure herein describes the manufacture, characterization, and use of molecules that bind hepatitis B virus (HBV) core (HBcAg) and e (HBeAg) antigens and thereby inhibit HBV infection and/or modulate a host immune system response. The molecules that bind to HBcAg and/or HBeAg, such as peptides, modified or derivatized peptides, peptidomimetics, and chemicals, are collectively referred to as xe2x80x9cbinding partnersxe2x80x9d. Binding partners can be obtained by synthesizing the heavy (VH) and light (VL) chains of antibodies (e.g., polyclonal, monoclonal, or fragments thereof), synthesizing the domains of proteins that interact with HBcAg and/or HBeAg, and by employing techniques in rational drug design and combinatorial chemistry.
Several synthetic peptides, derived from the variable domains of monoclonal antibodies (mAbs) specific for the hepatitis B virus HBcAg and/or HBeAg, were obtained as follows. The mnRNAs encoding the VH and VL chains of HBcAg and/or HBeAg monoclonal antibodies (mAbs) were sequenced and the protein sequences corresponding to these mRNAs were determined. Several synthetic peptides corresponding to these sequences were then synthesized using conventional protein chemistry. These xe2x80x9ccandidate binding partnersxe2x80x9d, which have the potential to bind HBcAg and/or HBeAg, were tested for the ability to interact with HBcAg and HBeAg. Five peptides, in particular, were discovered to bind HBcAg and/or HBeAg with high affinity and these xe2x80x9chigh affinityxe2x80x9d binding partners had either the conserved motif xe2x80x9cCKASxe2x80x9d (SEQ. ID. No. 77) or xe2x80x9cCRASxe2x80x9d (SEQ. ID. No. 78). Thus, preferred embodiments include peptides, derivative or modified peptides, or peptidomimetics having the formula xe2x80x9cX1nCKASX2nxe2x80x9d or xe2x80x9cX1nCRASX2nxe2x80x9d, wherein xe2x80x9cX2xe2x80x9d and xe2x80x9cX2xe2x80x9d are any amino acid and xe2x80x9cnxe2x80x9d is an integer, that bind HBcAg and/or HBeAg. Another way of describing the molecules of this class is by the formula: xe2x80x9cX1nCZASX2nxe2x80x9d, wherein: xe2x80x9cX1xe2x80x9d and xe2x80x9cX2xe2x80x9d are any amino acid and xe2x80x9cnxe2x80x9d is any integer, xe2x80x9cCxe2x80x9d is cysteine, xe2x80x9cZxe2x80x9d is lysine or argininexe2x80x9d, xe2x80x9cAxe2x80x9d is alanine, and xe2x80x9cSxe2x80x9d is serine.
By a similar approach, synthetic peptides corresponding to the binding domains of polyclonal antibodies specific for HIBcAg and/or HBeAg can be manufactured. Polyclonal antibodies specific for HBcAg and/or HBeAg are generated by inoculating animals with HBcAg and/or HBeAg. The mRNAs encoding the polyclonal antibodies are isolated, sequenced, and the protein sequences corresponding to these mRNAs are determined. Synthetic peptides corresponding to these protein sequences are then made using conventional techniques in protein chemistry. Several strategies for obtaining the imRNAs that encode polyclonal antibodies that bind HBcAg and/or HBeAg are contemplated including, but not limited to, yeast one-hybrid screens, yeast two-hybrid screens, and phage display techniques. Ideally, cDNA expression libraries corresponding to mRNAs encoding polyclonal antibodies that bind HBcAg and/or HBeAg are created. Embodiments that employ such libraries can express recombinant binding partners, which can be isolated or purified, characterized, and used in lieu of or in addition to synthetic binding partners.
The term xe2x80x9cisolatedxe2x80x9d requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polypeptide present in a living animal is not isolated, but the same polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. It is also advantageous that the sequences be in purified form. The term xe2x80x9cpurifiedxe2x80x9d does not require absolute purity; rather, it is intended as a relative definition. Isolated proteins have been conventionally purified to electrophoretic homogeneity by Coomassie staining, for example. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
In addition to antibodies and peptides derived from antibodies, other molecules that bind HBcAg and/or HBeAg can be identified by the methods described herein. That is, techniques in high throughput screening, combinatorial chemistry, and rational drug design can be employed to identify more binding partners. By one approach, for example, a high throughput screen based on a yeast two hybrid system is employed.
Accordingly, cDNA expression libraries are generated from any organism (e.g., plant, bacteria, virus, insect, amphibian, reptile, bird, or mammal) or a plurality of such organisms or from random or directed oligonucleotide synthesis. In some embodiments, the organism will have been immunized for HBcAg and/or HBeAg prior to creation of the cDNA expression library. To create the cDNA library for the yeast two-hybrid screen, isolated cDNA made from total mRNA obtained from the organism or generated by oligonucleotide synthesis is cloned into a first expression construct having a nucleic acid encoding a transcriptional activation domain (e.g., GAL4). As one of skill will appreciate, the cloning of the first expression construct is conducted such that cells having the construct will express a fusion protein comprising the cDNA of interest and the transcriptional activation domain when induced.
Next, a second expression construct (referred to as the xe2x80x9cbaitxe2x80x9d) is made. This construct has nucleic acid encoding HBcAg and/or HBeAg or fragments thereof joined to a transcriptional binding domain (e.g., GAL4). When a cell having the second expression construct is properly induced, a second fusion protein comprising the HBcAg and/or HBeAg or fragments thereofjoined to the transcriptional binding domain (the xe2x80x9cbaitxe2x80x9d) is expressed. The two expression constructs are transferred into yeast, which harbor a DNA template having at least one DNA binding domain specific for the transcriptional binding domain encoded by the bait construct, a minimal promoter, and a downstream reporter gene (e.g., Lac Z or Green Fluorescent Protein (GFP)). When a peptide from the cDNA library (i.e., the fusion protein expressed from the first construct) binds to the bait (i.e., the fusion protein from the second construct) a detectable signal is generated from the reporter gene. The yeast clones can be presented in addressable arrays, which allows for the precise determination of the clone containing an insert that encodes a protein that binds HBcAg and/or HBeAg. In this manner, protein/protein interactions between HBcAg and/or HBeAg and the proteins expressed from the cDNA library can be rapidly identified.
Clones that display a signal after induction of the first and second constructs but fail to produce a signal without induction are isolated, amplified, and the cDNA inserts are sequenced. The nucleic acid sequence information can be converted to an amino acid sequence and peptides corresponding to these sequences can be synthesized by conventional protein chemistry. Alternatively, recombinant peptides expressed from the positive clones can be isolated and/or purified. These candidate binding partners can then be screened for the ability to interact with HBcAg and/or HBeAg. The binding partners identified by the approaches above can also be used as templates for the design of modified or derivative peptides, peptidomimetics, and for rational drug design. For example, computer modeling and combinatorial chemistry can be employed to design and manufacture derivative binding partners.
The term xe2x80x9cbinding partnerxe2x80x9d also refers to a bi-functional binding partner or xe2x80x9cspecificity exchangerxe2x80x9d comprising a xe2x80x9cspecificity domainxe2x80x9d, which binds HBcAg and/or HBeAg, and an xe2x80x9cantigenic domainxe2x80x9d, which binds an antibody or other molecule that can be unrelated to a molecule that binds HBcAg and/or HBeAg. These bi-functional binding partners can redirect antibodies that already exist in an organism to a desired antigen. Such specificty exchangers can be manufactured by joining a molecule that binds HBcAg and/or HBeAg, such as a binding partner identified by a method described above, to an epitope for any antibody using conventional techniques in molecular biology. In one embodiment, for example, a specificity domain comprising the CKAS (SEQ. ID. No. 77) motif was joined to an antigenic domain comprising the epitope for a monoclonal antibody specific for the herpes simplex virus type 1 gG2 (HSVgG2) protein.
Desirably, the binding partners are evaluated in a xe2x80x9ccharacterization assayxe2x80x9d, which determines the ability of the molecule to interact with HBcAg and/or HBeAg, inhibit HBV infectivity, or modulate (inhibit or enhance) a host immune system response. Several characterization assays described herein involve binding assays that analyze whether a binding partner can interact with HBcAg and/or HBeAg and to what extent a binding partner can compete with other ligands for HBcAg and/or HBeAg (e.g., multimeric support-based assays and computer generated binding assays). Additionally, some characterization assays determine the efficacy of binding partners as inhibitors of HBV infection in vitro and in vivo. Further, characterization assays are designed to analyze whether a binding partner can modulate a host immune system response, as indicated by the activation of an antigen presenting cell (e.g., a B cell or dendritic cell), production of a cytokine, or T cell proliferation.
The binding partners can be used as biotechnological tools, diagnostic reagents, and the active ingredients in pharmaceuticals. In some embodiments, for example, the binding partners are used as detection reagents in conventional immunohistochemical techniques. In other embodiments, the binding partners are expressed in a cell in vitro or in vivo. Still in other embodiments, the binding partners are used as diagnostic reagents to detect the presence or absence of HBV in a biological sample obtained from a subject. According to this later aspect, the binding partners can also be used to determine the efficacy of an HBV treatment protocol by monitoring the levels of HBcAg and/or HBeAg before, during, and after treatment.
Further, binding partners can be incorporated into pharmaceuticals that can be administered to subjects in need of an agent that interacts with HBcAg and/or HBeAg, such as a human in need of treatment and/or prevention of HBV infection. Preferably, these pharmaceuticals comprise formulations having a specificity exchanger that promotes rapid clearance of HBV particles. Additionally, the pharmaceuticals can include nucleic acid constructs manufactured such that binding partners (preferably specificity exchangers) are expressed in a variety of cells of the body. The pharmaceuticals can be administered to individuals in need of treatment and/or prevention of HBV infection. The section below describes several approaches to identify and manufacture binding partners specific for HBcAg and/or HBeAg.
Identification and manufacture of binding partners specific for HBcAg and/or HBeAg
In general, the approach to make the binding partners described herein involves: (1) obtaining molecules that bind to HBcAg and/or HBeAg; (2) determining the molecular structure or sequence of said molecules; and (3) synthesizing peptides that have said molecular structure or sequence. In one aspect, for example, antibodies or other peptides that bind to HBcAg and/or HBeAg are generated and/or identified; the mRNA sequence encoding the binding partner is obtained, converted to cDNA, and sequenced; and, from this sequence, peptides are synthesized. The HBcAg and HBeAg-specific peptides can be modified, derivatized, and can also be used as templates for the design of peptidomimetics and rational drug discovery. Through techniques in combinatorial chemistry and rational drug design, many more binding partners can be identified. The term xe2x80x9cbinding partnerxe2x80x9d refers to a molecule that binds HBcAg and/or HBeAg, and should be distinguished from the term xe2x80x9ccandidate binding partnerxe2x80x9d, which refers to a molecule that potentially binds to HBcAg and/or HBeAg. Desirably, binding partners inhibit viral infectivity and/or modulate (inhibit or enhance) a host immune system response (e.g., antigen presenting cell activation, cytokine production, and/or T cell proliferation).
By one approach, the design and manufacture of peptides that bind HBcAg and HBeAg involves the manufacture of mAbs directed to HBcAg and HBeAg. Depending on the context, the term xe2x80x9cantibodiesxe2x80x9d can encompass polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. Furthermore, the terms xe2x80x9clow titer antibodyxe2x80x9d and xe2x80x9chigh titer antibodyxe2x80x9d are also used to refer to an antibody having a low avidity to an antigen and a high avidity to an antigen, respectively. That is, whether a particular antibody is a xe2x80x9clow titer antibodyxe2x80x9d or a xe2x80x9chigh titer antibodyxe2x80x9d depends on the dilution of antibody containing sera at which an antigen is no longer detectable in an enzyme immunoassay (e.g., an enzyme immunoassay (EIA) or ELISA assay); 200 ng of target antigen is typically used with a 1:1000 dilution of secondary antibody. Thus, a xe2x80x9clow titer antibodyxe2x80x9d generally no longer detects an antigen at a dilution that is less than 1:10000 under the conditions for ELISA described above and a xe2x80x9chigh titer antibodyxe2x80x9d is characterized by the ability to detect an antigen at a dilution that is greater than or equal to 1:10000.
For the production of antibodies, whether monoclonal or polyclonal, various hosts including goats, rabbits, rats, mice, etc. can be immunized by injection with HBcAg and/or HBeAg or any portion, fragment or oligopeptide that retains immunogenic properties. Depending on the host species, various adjuvants can be used to increase immunological response. Such adjuvants include, but are not limited to, Freund""s, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum are also potentially useful adjuvants.
Peptides used to induce specific antibodies can have an amino acid sequence consisting of at least three amino acids, and preferably at least 10 to 15 amino acids. Short stretches of amino acids encoding fragments of HBcAg and/or HBeAg can be fused with those of another protein such as keyhole limpet hemocyanin such that an antibody is produced against the chimeric molecule. While antibodies capable of specifically recognizing HBcAg and/or BBeAg can be generated by injecting synthetic 3-mer, 10-mer, and 15-mer peptides that correspond to a protein sequence of a binding partner into an appropriate organism, a more diverse set of antibodies are generated by using recombinant HBcAg and/or HBeAg.
Monoclonal antibodies directed to HBcAg and/or HBeAg can be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (Nature 256:495-497 (1975)), the human B-cell hybridoma technique (Kosbor et al. Immunol Today 4:72 (1983); Cote et al Proc Natl Acad Sci 80:2026-2030 (1983), and the EBV-hybridoma technique Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc, New York N.Y., pp 77-96 (1985)). In addition, techniques developed for the production of xe2x80x9cchimeric antibodiesxe2x80x9d, the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used. (Morrison et al. Proc Natl Acad Sci 81:6851-6855 (1984); Neuberger et al. Nature 312:604-608(1984); Takeda et al. Nature 314:452-454(1985)). Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce HBcAg and/or HBeAg-specific single chain antibodies. Antibodies can also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al., Proc Natl Acad Sci 86: 3833-3837 (1989), and Winter G. and Milstein C; Nature 349:293-299 (1991).
Antibody fragments that contain specific binding sites for HBcAg and/or HBeAg can also be generated. For example, such fragments include, but are not limited to, the F(abxe2x80x2)2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(abxe2x80x2)2 fragments. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (Huse W. D. et al. Science 256:1275-1281 (1989)).
By one approach, monoclonal antibodies to HBcAg and/or HBeAg or fragments thereof are made as follows. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein or peptides derived therefrom over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused in the presence of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution are placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall, E., Meth. Enzymol. 70:419 (1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L. et al. Basic Methods in Molecular Biology Elsevier, N.Y. Section 21-2. By using the approach described in Example 1, several monoclonal antibodies specific for HBcAg and/or HBeAg were made.